CA1177088A - Method for the extraction of silymarin from plants - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Silymarin (silymarin I, II, III and IV) is produced in a purity superior to that of prior methods in a new method in which a large part of the oil is removed from the fruits of Silybum marianum by cold pressing, the compressed mass is broken up, the pressed residue is extracted with anhydrous ethyl acetate and the ethyl acetate extract is evaporated, the residue therefrom is defatted, a methanolic solution of the defatted residue is separated with a chlorine substituted lower alkane, and silymarin is recovered from the aqueous methanolic phase.

Description

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The invention concerns a method for the isolation of pure silymarin by extraction from the fruits of the milk thistle (Silybum marianum Gaertn.).
In technical usagé the term silymarin is used to denote the silymarin group, namely the polyhydroxyphenyl-chromanones (silymarin I, II, III and IV) which can be isolated from the fruits of the milk thistle with 70to 80%
purity, depending on the quality of the crude drug ~West German Patent (Auslegeschrift) 1 923 082).
Silymarin I, silymarin II and silymarin III are isomers having the empirical formula C25H22Olo. In the literature dealing with their structural formulae they have been given the names silybin, silydianin and silychristin, ~West German Patent 1 923 082; PELTER, A. and HANSEL, R
Tetrahedron Letters, London 25, 2911, 1968, WAGNER, H., HORHAMMER, L., SELIGMANN. O., and F~RNSWORT~, N. R., Tetra-hedron ~etters ~o. 31, pages 2675 - 2678, 1970, WAGNER, H., SELIGMANN, O., HORHAMMER, L. and SEITZ, M., Tetrahedron Letters No. 22, pages 1895 - 1899, 1971)~ Silymarin IV is taxifolin (German Patent 1 923 082).
Silymarin is a valuable agent for the therapy of liver diseases, and the silymarin mixture, by virtue of synergism, has a greater effect than the sum of the effects of the separate isomers (West German Patent 1 923 082).
A method for the isolation of silymarin (silymarin I - IV) is known from West German Patent 1 923 082. In this method the dried seeds of Silyburn marianum Gaertn. are mecha-nically pressed so as to rid them of the major part of the fatty oil. The pressed residue is then thoroughly extracted with ethyl acetate, the ethyl acetate solution is evaporated, the dry residue is redissolved in anhydrous methanol, the '~

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solution is defatted by repeated uniform countercurrent partition, and the fat-~ree methanolic solution is evaporated to dryness in vacuo~ This gives a 70 to 80% polyhydroxyphenyl-chromanone mixture (silymarin I - IV). The proportions of silymarin I, II and III are approximate]y 3:1:1. The amount of silymarin IV (taxifolin) is approximately 2 to 3% of the total silyrnarin content.
West German Offenlegungsschrift 2 020 407 represents a further technical advance. The procedure set out in this publication deals with the isolation of silymarin without preliminary defatting. It is based on direct overall extraction with a "solvent mixture of alcohol-water-a lower fatty acid ester or a chlorinated hydrocarbon" and separation of the fatty constituents "by subsequently distilling off 10 to 40~/O
of the volume of the resulting extract solu-tion, leaving it to stand and then removing the fats which rise to the surface of the solution". According to the publication, this method yields a 40/O silymarin extract' the publication recommends recrystallization from methanol, without giving any further information regarding appropriate technical measures. On attempting to carry out this procedure the promised yield proved not to be reproducible, and approximately 13% of the original fat was still present in the end productO
A further method is described in West German Offenlegungsschrift 2 017 789. The end products of this method are claimed to contain 40 to 70/O of silymarin, and the yield of end product ranges from 0.3/O to a maximum of 2.4%
(of 40/O silymarin), A practical trial showed that this method, too, was not reproducible.

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A further contribution is to be found in French Patent No. 72.14042, which concerns the production of an ex~ract from Silybum marianum without preliminary de~atting.
It is claimed that the end product contains approximately 82%
of polyhydroxyphenylchromones, silymarins are stated to constitute 50~/O of this 82%. It may be noted that silymarin belongs to a different class of compounds, namely the poly-hydroxyphenylchromanones and not the polyhydroxyphenylchromones.
This method has also been found to be not reproducible.
It has so ~ar proved impossible to make any sub-stantial improvement on the method set out in West German Patent 1 923 082. Difficulties arise primarily in separating the active constituents from the crude drug while preserving the mixture of silymarins present in it, and secondly in separating this mixture from approximately 3~/O of fa-tty oils and other incidental substances which are not easily removed.
These difficulties have only been partially overcome by West German Patent 1 923 082.
As it is a valuable agent for the treatment of liver disease, there is a demand for sil~marin in the highest possible concentration, indeed in concentrations higher than those available in the present state of technical progress.
The purpose of the present invention is to devise a new process for the isolation of pure silymarin (silymarin I - IV) substantially free from incidental substances and containing all the natural isomers. This new process is intended to lead along a simple pathway to a higher yield of an outstandingly pure product.
Surprisingly enough, it has proved possible to devise a process which avoids the disadvantages of the previous methods, which can be operated economically and which yields 7~
pure silymarin (silymarin I - I~) (purity 90 to g6%)~ measur-ed according to the method of Wagner, Horharnmer, Seligmann and Farnsworth.
This invention concerns a method for isolating silymarin (silymarin I - IV) according to ~hich the fruits of silybum marianum are freed from a large proportion of their fatty oil by cold pressing, the pressed mass is pulverized, the pressed residue extracted with ethyl acetate, and the ethyl acetate extract is evaporated and further processed.
In particular the method of the invention suitably employs the following steps:
a) the pressed residue is extracted several times with anhydrous ethyl acetate and the extract separated from the crude drug residue, b) the residue left after evaporating the combined ethyl acetate extracts is defatted by repeated solid-liquid extraction with light petroleum or, bl) the residue remaining after evaporation is dissolved in methanol and the solution is defatted by repeated liquid-liquid extraction with light petroleum, c) as much as possible of the defatted residue b) is dissolved in methanol and the solution separated from the undissolved fraction or, c') the defatted methanolic solution b') is separated from the light petroleum, d) the methanolic solution is mixed with a chlorinated lower alkane as solvent to~ether with water. The mixture is shaken, and the aqueous-methanolic solution is separated and retained, e) the chlorinated solvent phase is extracted several times by stirring with methanol and water and decanted, '7~

f) the combined a~ueous-methanolic phases are evaporated, g) the residue left on evaporation is suspended in a little methanol, the suspension is then stirred into water and after centrifuging the precipitate is washed several times with water, h) the precipitate is dried in vacuo, pulverized and again dried in vacuo.
A particularly preferred embodiment of the inven-tion employs the following steps:
a) the pressed residue is extracted from twice to four times, preferably three times, for 1/2 to 1-1/2 hours, preferably 1 hour, with anhydrous ethyl acetate at 65 to 76C, preferably 75C, in the proportions 1:8 to 1:12, preferably 1:10, with vigorous stirring and the extracted crude drug is removed, b) the combined ethyl acetate extracts are evaporated at 40 to 55C, preferably 50C, in vacuo at 15 mbar, c) the residue left on evaporation is defatted by solid-liquid extraction with light petroleum 60/70 in the proportions 1:15 to 1:25, preferably 1:20. This extraction is carried out three times for periods of 10 to 20minuteS~ preferably 15 minutes, d) the defatted residue is dissolved in 6 to 10 times, preferably 8 times, its weight of methanol and the undissolved fraction is removed, e) chloroform and water, each in volumes equal to that of methanolic solution, are added to the latter and the mixture is vigorously stirred or 15 minutes and decanted, f) the chloroform phase is mixed with methanol and water 7'7~

in proportions by volume 1:1:1 and stirred vi~orously for 15 minutes. This process is carried out five times in all, the chloroform phase being removed at the end of each extraction, g) the combined aqueous-methanolic phases are evaporated at 30 to 50C, preferably 40C, in vacuo at 15 mbar, h) the residue left on evaporation is suspended in methanol in the proportions 1:4 to 1:6, preferably 1:5, and the suspension is stirréd into 8 to 12 times, preferably 10 times, its volume of water. The preci-pitate is separated by centrifugation and washed repeatedly with water, i) the precipitate is dried for 20 to 30 hours, prefer-ably 24 hours, at 30 to 50C, preferably 40C, in vacuo at 10 mbar. It is then pulverized and the pulverized product is again dried at 30 to 50C, preferably 40C, in vacuo at 10 mbar.
The products isolated in this way have a silymarin (silymarin I - IV) content of 90 to 96%, calculated in terms of the anhydrous substance.
As regards production technology, the individual steps and the specific procedures have been selected so as to ensure that the mixture of active substances present in the crude drug is almost completely recovered. Deviation from these procedures results in partial or total loss of one or other of the compounds~ This difficulty assumes grow-ing proportions as the concentration of the mixture of active substances rises, especially during the later stages of the method, because the active substances tend to be lost, either as individual compounds or as the mixture, with the incidental substances from which they have to be separated.

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The method which is the subject of this invention has been devised with due attention to these points, so that it g~ves an optimal yield of a pure product containing the isomers in substantially the same proportions as in the crude drug.
It is a remarkable fact that only by a carefully chosen combination of production techniques, solvents of solvent systems and physical or chemical conditions that optimal product can be obtained.
By subjecting the fruits of Silybum marianum to cold pressing it is possible to prevent any chemical break-down or polymerization of the silymarin and to avoid an intimate emulsification of the silymarin and the incidental substances in the fatty oil, an event which would hinder their subsequent separation. The crude drug is partially defatted by the pressing,approximately 25 to 30% of the oil heing expelled.
The fine pulverization step speeds up the subsequent extraction, because it allows the solvent to penetrate more 20 easily into the crude drug particles~
One critical feature of the process of the invention is that anhydrous ethyl acetate is used for the ethyl acetate extraction. The maximum permissible water content is 0.5%.
If the water content exceeds 0.5%, it will be impossible to separate the incidental substances during the subsequent steps in the procedure. This remarkable effect, taken in combination - with the other technical procedures, is vital for the success of the process, namely the isolation of pure silymarin.
The ratio of crude drug to solvent during the ethyl 30 acetate extraction is suitably 1:8 to 1:12, preferably 1:10.

This ratio has been selected so as to ensure that the silymarin is specifically and safely dissolved, without any unnecessary excess of solvent. By repeating the extraction twice to four times, preferably three times, complete removal oE the sily-marin from the crude drug is guaranteed. The working tempera-ture of 60 to 80C, preferably 75C, favours complete solution of the active substances while at the same time avoiding any degradation of the chemical compounds present in the drug.
Stirring with an intensive stirrer causes mechanical disintegration of the crude drug, a change which facilitates extraction. The short time of 1/2 to 1-1/2 hours, preferably 1 hour, chosen for each of the extraction steps nevertheless ensures that the extraction is complète.
The combined ethyl acetate extracts are evaporated at low temperature in vacuo in such a way as to avoid any risk of degrading the active substance.
The next step is repeated solid-liquid defatting of the residue with light petroleum using a relatively high proportion of residue to light petroleum, for example about 1:20. This has the advantage that the fatty oil can be completely removed in a short time and without elaborate apparatus as compared for example with an elaborate liquid-liquid countercurrent extraction process in the present state of technical development.
Furthermore, the solid-liquid separation process avoids any loss of active substances, as often occurs during liquid-liquid extraction, because the solvent used for purification emulsifies or dissolves a proportion of the solvent containing the active substance.
Methanol, a relatively inexpensive solvent, is par-ticularly suitable for redissolving the defa~ted residue by virtue of its powerful solvation properties. Silymarin ~silymarin I - IV) is easily soluble in methanol. The high proportion of residue to solvent has been chosen to ensure that the silymarin ~silymarin I - IV) shall be completely redissolved. No silymarin remains in the resi~ue.
One new and particularly ingenious feature is the use of chlorinated organic solvents, in particular chlorine substituted lower alkanes, for example chloroform, methylene chloride, 1,2-dichloroethane or trichlorethylene, preferably chloroform, for separating the remaining indicidental substances from the silymarin (silymarin I - IV).
This ensures that the silymarin as a whole undergoes no loss and that none of the isomers escapes. This procedure eliminates further lipophilic incidental substances which were not removed during the defatting step together with the oil and light petroleum, and also other less strongly polar incidental substances, which were at first dissolved in the methanol but which behave appropriately in a partition phase system~ The solution equilibrium (phase diagram) of the selectedsolvent system, both in its nature and its quantity, in this case methanol-water-chloroform 1:1:1, is particularly suitable for silymarin (silymarin I - IV) and any incidental substances which may still be present, because good separation into two phases can be achieved with this solvent system~
The dissociation zone of the phase diagram comprises the stated solvent proportions, The partition coefficient of silymarin (silymarin I - IV) between the two phases is very high. After carrying out six extractions by shaking in a system consisting of methanol/chloroform~water 1/1/1 (v/v/v) the proportion of silymarin in the combined aqueous-methanol phases to that remaining in the chloroform phase is as 13:1~

_ g _ In this way the silymarin ~silymarin I - V) can be almost comple-tely extracted from the methanolic phase.
One remarkable result obtained by this invention is that silymarin I to IV remain in their due proportions as individual compounds in the aqueous-methanolic phase, so that the ratio of the isomers is preserved.
The aqueous-methanolic phase is evaporated to dry-ness in vacuo at low temperature with due precautions to~
prevent decomposition and the residue is suspended in water for repeated washing. This washing process has proved advan-tageous in that it overcomes the difficulties caused by blocking of the filters by impurities in the product during ordinary filtration processes.
As opposed to the methods available in the present state of technical development, this invention has the advanta~es of being simple and of producing 90 to 96% pure silymarin (silymarin I - IV) in a yield of approximately 1.7%. Only by employing the selected procedures which are the subject of this invention, namely the use of anhydrous ethyl acetate and a chlorine substituted alkane, for ~xample chloroform, is it possible to isolate substantially pure silymarin from the fruits of Silybum marianum.
The effect of the extraction of the drug with -anhydrous ethyl acetate is especially inventive compared with the technical status. Hydrophilic materials in the drug remain in the residue. During the following processes, such as distribution in solvents, these hydrophilic accompanying materials cannot normally be completely separated from the silymarin.
Furthermore, the efficiency of distribution of the components in solvent systems methanol ~ chloroform or '7~

methanol / chlorine-substituted lower alkane is especially surprising. The distribution of the components is speci~i-cally selective, that is, the methanolic phase contains the active ingredient silymarin almost exclusively, whereas the chloroform phase contains practically all the accompanying materials.
The invention is illustrated in particular and preferred embodiments in the following examples:

300 kg fi~ely pulverized, cold-pressed milk thistle fruits are mixed with 3000 1 anhydrous ethyl acetate and heated to 75 C.
At this temperature the mixture is vigorously stirred with an intensive stirrer for one hour. It is then filtered and the crude drug residue is extracted twice more with ethyl acetate, as described.
The combined, clarified ethyl acetate solutions are evaporated to dryness at 50C in vacuo at 15 mbar. For the purpose of defatting, the residue is stirred three times with 300 1 quantities of light petroleum 60/70C at room tempera-ture for 15 minutes and then freed from solvent by dryiny in vacuo. The crude product thus obtained is redissolved in four times its volume of methanol. The undissolved solid material is separated by centrifugation, washed twice with half the above mentioned quantity of methanol and finally, as it now contains practically no silymarin, discarded.
The combined methanolic solutions are mixed first with chloroform and then with water in proportions by volume ~ 1 and transferred to a decanting vessel.
The mixture is then thoroughly stirred for 15 minu-tes and the phases are allowed to separate. The lower phase ~1'i"7~

(the chloroform phase) is then drawn off and extrac-ted with methanol-water in proportions by volume 1:1:1 for 15 minutes~
This extraction is repeated five times in all. The chloro-form phase is then discarded.
The combined aqueous-methanolic phases are evapo-rated in vacuo at 15 mbar at 50C and the residue is sus-pended in five times its volume of methanol. The suspension is then added in a thin stream to ten times its volume of water with continuous stirring, which is then continued vigorously for a further 15 minutes. The mixture is then , centrifuged and the solid material is washed by resuspending it in water several times and recentrifuging. The product is dried in a vacuum oven at 40C for 24 hours, pulverized and again dried at 40C for a further 48 hours.
The result is a light beige powder which has a silymarin content of 90 to 96% (determined by the dinitro-phenylhydrazine method (DNP~ method) as used for the deter-mination of silymarin in milk thistle fruits, DAB VIIth edition 1968, 2nd supplement 1965, page 198).
Identification is carried out by thin layer chromotography.
The proportions of the individual isomers are determined by quantitative evaluation of the remission curves.
The remission curves are shown graphi~ally in Figure 1 of the accompanying drawings.
With further reference to Figure 1 plot 1 is silybin (silymarin I), plot 2 is silydianin (silymarin II), plot 3 is silychristin (silymarin III) and plot 4 is taxifolin (silymarin IV). The plots were determined at a wave length of 288 nm.

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The yield amounts to 4.9 kg, equivalent to 1.7%
of the crude drug in dry form.

The procedure of Example 1 was carried out but employing methylene chloride instead of chloroform, and there was recovered a product containing 90 to 96% of silymarin.
The yield amounts to 1.7%.
EXAMPI,E 3 The procedure of Example 1 was carried out but employing 1l2-dichloroethane instead of chloroform, and there was recovered a product containing 90 to 9~/O of silymarin.
The yield amounts to 1.7%.

The procedure of Example 1 was carried out but employing trichlorethylene instead of chloroform and there was recovered a product containing 90 to 94% of silymarin.
The yield amounts to 1. 8%, In all the examples the percentage purity and little variations concerning the yield depend on the drug~ The percentages are by weight unless otherwise indicated.

Claims (23)

The embodiments of the invention in which an exclu-sive property or privilege is claimed are defined as follows:

1. A method for the extraction of silymarin, in which a large part of the oil is removed from the fruits of Silybum marianum by cold pressing, the compressed mass is broken up, the pressed residue is extracted with anhydrous ethyl acetate and the ethyl acetate extract is evaporated, the residue therefrom is defatted, a methanolic solution of the defatted residue is separated with a chlorine substituted lower alkane and silymarin is recovered from an aqueous methanolic phase.

2. A method according to claim 1 wherein a) the pressed residue is extracted several times with anhydrous ethylacetate and the extract separated from the residue, b) the residue remaining after evaporation of the com-bined ethyl acetate extracts is defatted by repeated solid-liquid extraction with light petroleum, c) the greater part of the defatted residue b) is dissolved in methanol and the solution separated from the undissolved fraction, d) to the methanolic solution is added a chlorine-sub-stituted lower alkane as solvent together with water, after shaking, the aqueous-methanolic solution is separated and retained, e) the chlorine-substituted solvent phase is extracted several times by stirring with methanol and water and decanted, f) the combined aqueous-methanolic phases are evaporated, g) the residue left on evaporation is suspended in a small amount of methanol, the suspension is then stirred into water and after centrifugation the deposit is washed several times with water, h) the deposit is dried in vacuo, pulverized and again dried in vacuo.

3. A method according to claim 1 wherein a) the pressed residue is extracted several times with anhydrous ethyl acetate and the extract separated from the residue, b) the residue remaining after evaporation is dissolved in methanol and the solution is defatted by repeated liquid-liquid extraction with light petroleum, c) the defatted methanolic solution is separated from the light petroleum, d) to the methanolic solution is added a chlorine-sub-stituted lower alkane as solvent together with water after shaking, the aqueous-methanolic solution is separated and retained, e) the chlorine-substituted solvent phase is extracted several times by stirring with methanol and water and decanted, f) the combined aqueous-methanolic phases are evaporated, g) the residue left on evaporation is suspended in a small amount of methanol, the suspension is then stirred into water and after centrifugation the deposit is washed several times with water, h) the deposit is dried in vacuo, pulverized and again dried in vacuo.

4. A method according to claim 1 wherein a) the pressed residue is extracted from twice to four times, for 1/2 to 1-1/2 hours, with anhydrous ethyl acetate at 65 to 76°C, in the proportions 1:8 to 1:12, with vigorous stirring and the extracted crude drug is removed, b) the combined ethyl acetate extracts are evaporated at 40 to 55°C, in vacuo at 15 mbar, c) the residue left on evaporation is defatted by solid-liquid extraction with light petroleum 60/70° in the proportions 1:15 to 1:25, said extraction being carried out three times for periods of 10 to 20 minutes, d) the defatted residue is dissolved in 6 to 10 times, its weight of methanol and the undissolved fraction is removed, e) chloroform and water, each in volumes equal to that of the methanolic solution, are added to the latter and the mixture is vigorously stirred for 15 minutes and decanted, f) the chloroform phase is mixed with methanol and water in proportions by volume 1:1:1 and stirred vigorously for 15 minutes, this step being carried out five times in all, the chloroform phase being removed at the end of each extraction, g) the combined aqueous-methanolic phases are evaporated at 30 to 50°C, in vacuo at 15 mbar, h) the residue left on evaporation is suspended in methanol in the proportions 1:4 to 1:6, and the suspension is stirred into 8 to 12 times, its volume of water, the precipitate therefrom being separated by centrifugation and washed repeatedly with water, i) the precipitate is dried for 20 to 30 hours, at 30 to 50°C, in vacuo at a0 mbar, and is then pulverized and the pulverized product is again dried at 30 to 50°C, in vacuo at 10 mbar.

5. A method according to claim 1 wherein a) the pressed residue is extracted three times, for 1 hour with anhydrous ethyl acetate at 75°C in the proportions 1:10 with vigorous stirring and the extracted crude drug is removed, b) the combined ethyl acetate extracts are evaporated at 50°C in vacuo at 15 mbar, c) the residue left on evaporation is defatted by solid-liquid extraction with light petroleum 60/70° in the proportions 1:20, said extraction being carried out three times for periods of 15 minutes, d) the defatted residue is dissolved in 8 times its weight of methanol and the undissolved fraction is removed, e) chloroform and water, each in volumes equal to that of the methanolic solution, are added to the latter and the mixture is vigorously stirred for 15 minutes and decanted, f) the chloroform phase is mixed with methanol and water in proportions by volume 1:1:1 and stirred vigorously for 15 minutes, this step being carried out five times in all, the chloroform phase being removed at the end of each extraction, g) the combined aqueous-methanolic phases are evaporated at 40°C in vacuo at 15 mbar, h) the residue left on evaporation is suspended in methanol in the proportion 1:5 and the suspension is stirred into 10 times its volume of water, the precipitate therefrom being separated by centrifuga-tion and washed repeatedly with water, i) the precipitate is dried for 24 hours at 40°C in vacuo at a0 mbar, and is then pulverized and the pulverized product is again dried at 40°C in vacuo at 10 mbar.

6. A method according to claim 1 wherein said silymarin is in a purity of at least 90%, by weight.

7. A method according to claim 2 wherein said silymarin is recovered in a purity of at least 90%, by weight.

8. A method according to claim 3 wherein said silymarin is recovered in a purity of at least 90%, by weight.

9. A method according to claim 4 wherein said silymarin is recovered in a purity of at least 90%, by weight.

10. A method according to claim 5 wherein said silymarin is recovered in a purity of at least 90%, by weight.

11. A method according to claim 1 wherein said silymarin is recovered as a product having a silymarin content of 90 to 96%, by weight, calculated as anhydrous substance.

12. A method according to claim 2 wherein said silymarin is recovered as a product having a silymarin content of 90 to 96%, by weight, calculated as anhydrous substance.

13. A method according to claim 3 wherein said silymarin is recovered as a product having a silymarin content of 90 to 96%, by weight, calculated as anhydrous substance.

14. A method according to claim 4 wherein said silymarin is recovered as a product having a silymarin content of 90 to 96%, by weight, calculated as anhydrous substance.

15. A method according to claim 5 wherein said silymarin is recovered as a product having a silymarin content of 90 to 96%, by weight, calculated as anhydrous substance.

16. Silymarin having a percentage purity of at least 90%, by weight, whenever prepared by the process of claim 6, 7 or 8 or by an obvious chemical equivalent.

17. Silymarin having a percentage purity of at least 90%, by weight, whenever prepared by the process of claim 9 or 10 or by an obvious chemical equivalent.

18. A product having a silymarin content of 90 to 96%, by weight, calculated as anhydrous substance whenever prepared by the process of claim 11, 12 or 13.

19. A product having a silymarin content of 90 to 96%, by weight, calculated as anhydrous substance whenever prepared by the process of claim 14 or 15.

20. An improved method for the extraction of silymarin from the fruits of Silybum marianum, the method being the type wherein a large part of the oil is removed from the fruits by cold pressing, the compressed mass is broken up, and the pressed residue extracted to obtain silymarin, and which comprises the steps of extracting the broken up compressed mass with anhydrous ethyl acetate to remove the silymarin therefrom, evaporating the ethyl acetate to leave a fatted residue; defatting the residue, extracting a methanolic solution of the defatted residue with a chlorine-substituted lower alkane, with the addition of water, extracting the chlorine-substituted solvent with a methanol-water solution; combining all of the methanolic solutions and evaporating the solvent to leave a product residue;
suspending the product residue in a small amount of methanol;
pouring the suspended residue into water, separating the methanol-water mixture from the material in suspension and drying the material in suspension to yield the desired product.

21. The method of claim 20 wherein the residue is defatted by extraction with a low boiling petroleum solvent.

22. The method of claim 21 wherein the residue is dissolved in methanol before extraction with the petroleum solvent.

23. The method of claim 20 wherein the broken up mass is extracted two to four times with anhydrous ethyl acetate at 65° to 76°C in a 1-8 to 1:12 proportion, the fatted residue is defatted by extraction with a low boiling petroleum solvent in a 1:15 to 1:25 ratio of residue to petroleum solvent, the defatted residue is dissolved in 6-10 times its weight of methanol, the methanol solution of defatted residue is extracted with chloroform as the chlorine-substituted lower alkane and water each in a volume about equal to that of the methanol solution, extracting the chloroform with methanol water is accomplished with a volume ration of about 1:1:1 of chloro-form:methanol:water.